The present invention relates to variants (mutants) of parent Termamyl-like xcex1-amylases with higher activity at medium temperatures and/or high pH.
xcex1-Amylases ( xcex1-1,4-glucan-4-glucanohydrolases, EC 3.2.1.1) constitute a group of enzymes which catalyze hydrolysis of starch and other linear and branched 1,4-glucosidic oligo- and polysaccharides.
There is a very extensive body of patent and scientific literature relating to this industrially very important class of enzymes. A number of xcex1-amylases such as Termamyl-like xcex1-amylases variants are known from e.g. WO 90/11352, WO 95/10603, WO 95/26397, WO 96/23873 and WO 96/23874.
Among more recent disclosures relating to xcex1-amylases, WO 96/23874 provides three-dimensional, X-ray crystal structural data for a Termamyl-like xcex1-amylase which consists of the 300 N-terminal amino acid residues of the B. amyloliquefaciens xcex1-amylase (BAN(trademark)) and amino acids 301-483 of the C-terminal end of the B. licheniformis xcex1-amylase comprising the amino acid sequence (the latter being available commercially under the tradename Termamyl(trademark)), and which is thus closely related to the industrially important Bacillus xcex1-amylases (which in the present context are embraced within the meaning of the term xe2x80x9cTermamyl-like xcex1-amylasesxe2x80x9d, and which include, inter alia, the B. licheniformis, B. amyloliquefaciens (BAN(trademark)) and B. stearothermophilus (BSG(trademark)) xcex1-amylases). WO 96/23874 further describes methodology for designing, on the basis of an analysis of the structure of a parent Termamyl-like xcex1-amylase, variants of the parent Termamyl-like xcex1-amylase which exhibit altered properties relative to the parent.
The present invention relates to novel xcex1-amylolytic variants(mutants) of a Termamyl-like xcex1-amylase which exhibit improved wash performance (relative to the parent xcex1-amaylase) at high pH and at a medium temperature.
The term xe2x80x9cmedium temperaturexe2x80x9d means in the context of the invention a temperature from 10xc2x0 C. to 60xc2x0 C. preferably 20xc2x0 C. to 50xc2x0 C., especially 30-40xc2x0 C.
The term xe2x80x9chigh pHxe2x80x9d means the alkaline pH which today are used for washing, more specifically from about pH 8 to 10.5.
In the context of the invention a xe2x80x9clow temperature xcex1-amylasexe2x80x9d means an xcex1-amylase which has an relative optimum activity in the temperature range from 0-30xc2x0 C.
In the context of the invention a xe2x80x9cmedium temperature xcex1-amylasexe2x80x9d means an xcex1-amylase which has an optimum activity in the temperature range from 30-60xc2x0 C. For instance, SP690 and SP722 xcex1-amaylases, respectively, are xe2x80x9cmedium temperature xcex1-amylases.
In the context of the invention a xe2x80x9chigh temperature xcex1-amylasexe2x80x9d is an xcex1-amylase having the optimum activity in the temperature range from 60-110xc2x0 C. For instance, Termamyl is a xe2x80x9chigh temperature xcex1-amylase.
Alterations in properties which may be achieved in variants(mutants) of the invention are alterations in: the stability of the Termamyl-like xcex1-amylase at a pH from 8 to 10.5, and/or the Ca2+ stability at pH 8 to 10.5, and/or the specific activity at temperatures from 10 to 60xc2x0 C., preferably 20-50xc2x0 C., especially 30-40xc2x0 C.
It should be noted that the relative temperature optimum often is dependent on the specific pH used. In other words the relative temperature optimum determined at, e.g., pH 8 may be substantially different from the relative temperature optimum determined at, e.g., pH 10.
The temperature""s influence on the enzymatic activity
The dynamics in the active site and surroundings are dependent on the temperature and the amino acid composition and of strong importance for the relative temperature optimum of an enzyme. By comparing the dynamics of medium and high temperature xcex1-amylases, regions of importance for the function of high temperature xcex1-amylases at medium temperatures can be determined. The temperature activity profile of the SP722 xcex1-amaylase (SEQ ID NO: 2) and the B. licheniformis xcex1-amylase (available from Novo Nordisk as Termamyl(copyright)) (SEQ ID NO: 4) are shown in FIG. 2.
The relative temperature optimum of SP722 in absolute activities are shown to be higher at medium range temperatures (30-60xc2x0 C.) than the homologous B. licheniformis xcex1-amylase, which have an optimum activity around 60-100xc2x0 C. The profiles are mainly dependent on the temperature stability and the dynamics of the active site residues and their surroundings. Further, the activity profiles are dependent on the pH used and the pKa of the active site residues.
In the first aspect the invention relates to a variant of a parent Termamyl-like xcex1-amylase, which variant has xcex1-amylase activity, said variant comprises one or more mutations corresponding to the following mutations in the amino acid sequence shown in SEQ ID NO: 2:
T141, K142, F143, D144, F145, P146, G147, R148, G149, Q174, R181, G182, D183, G184, K185, A186, W189, S193, N195, H107, K108, G109,D166, W167, D168, Q169, S170, R171, Q172, F173, F267, W268, K269, N270, D271, L272, G273, A274, L275, K311, E346, K385, G456, N457, K458, P459, G460, T461, V462, T463.
A variant of the invention have one or more of the following substitutions or deletions:
T141A,D,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,W,Y,V;
K142A,D,R,N,C,E,Q,G,H,I,L,M,F,P,S,T,W,Y,V;
F143A,D,R,N,C,E,Q,G,H,I,L,K,M,P,S,T,W,Y,V;
D144A,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;
F145A,D,R,N,C,E,Q,G,H,I,L,K,M,P,S,T,W,Y,V;
P146A,D,R,N,C,E,Q,G,H,I,L,K,M,F,S,T,W,Y,V;
G147A,D,R,N,C,E,Q,H,I,L,K,M,F,P,S,T,W,Y,V;
R148A,D,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;
G149A,D,R,N,C,E,Q,H,I,L,K,M,F,P,S,T,W,Y,V;
R181*,A,D,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;
G182*,A,D,R,N,C,E,Q,H,I,L,K,M,F,P,S,T,W,Y,V;
D183*,A,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;
G184*,A,R,D,N,C,E,Q,H,I,L,K,M,F,P,S,T,W,Y,V;
K185A,D,R,N,C,E,Q,G,H,I,L,M,F,P, S,T,W,Y,V;
A186D,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;
W189A,D,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,Y,V;
S193A,D,R,N,C,E,Q,G,H,I,L,K,M,F,P,T,W,Y,V;
N195A,D,R,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;
H107A,D,R,N,C,E,Q,G,HI,L,K,M,F,P,S,T,W,Y,V;
K108A,D,R,N,C,E,Q,G,H,I,L,M,F,P,S,T,W,Y,V;
G109A,D,R,N,C,E,Q,H,I,L,K,M,F,P,S,T,W,Y,V;
D166A,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;
W167A,D,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,Y,V;
D168A,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;
Q169A,D,R,N,C,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;
S170A,D,R,N,C,E,Q,G,H,I,L,K,M,F,P,T,W,Y,V;
R171A,D,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;
Q172A,D,R,N,C,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;
F173A,D,R,N,C,E,Q,G,H,I,L,K,M,P,S,T,W,Y,V;
Q174*,A,D,R,N,C,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;
F267A,D,R,N,C,E,Q,G,H,I,L,K,M,P,S,T,W,Y,V;
W268A,D,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,Y,V;
K269A,D,R,N,C,E,Q,G,H,I,L,M,F,P,S,T,W,Y,V;
N270A,D,R,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;
D271A,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;
L272A,D,R,N,C,E,Q,G,H,I,K,M,F,P,S,T,W,Y,V;
G273A,D,R,N,C,E,Q,H,I,L,K,M,F,P,S,T,W,Y,V;
A274D,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;
L275A,D,R,N,C,E,Q,G,H,I,K,M,F,P,S,T,W,Y,V;
K311A,D,R,N,C,E,Q,G,H,I,L,M,F,P,S,T,W,Y,V;
E346A,D,R,N,C,Q,G,H,I,K,L,M,F,P,S,T,W,Y,V;
K385A,D,R,N,C,E,Q,G,H,I,L,M,F,P,S,T,W,Y,V;
G456A,D,R,N,C,E,Q,H,I,L,K,M,F,P,S,T,W,Y,V;
N457A,D,R,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;
K458A,D,R,N,C,E,Q,G,H,I,L,M,F,P,S,T,W,Y,V;
P459A,D,R,N,C,E,Q,G,H,I,L,K,M,F,S,T,W,Y,V;
G460A,D,R,N,C,E,Q,H,I,L,K,M,F,P,S,T,W,Y,V;
T461A,D,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,W,Y,V;
V462A,D,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y;
T463A,D,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,W,Y,V.
Preferred are variants having one or more of the following substitutions or deletions:
K142R; S193P; N195F; K269R,Q; N270Y,R,D; K311R; E346Q; K385R; K458R; P459T; T461P; Q174*; R181Q,N,S; G182T,S,N; D183*; G184*; K185A,R,D,C,E,Q,G,H,I,L,M,N,F,P,S,T,W,Y,V; A186T,S,N,I,V,R; W189T,S,N,Q.
Especially preferred are variants having a deletion in positions D183 and G184 and further one or more of the following substitutions or deletions:
K142R; S193P; N195F; K269R,Q; N270Y,R,D; K311R; E346Q; K385R; K458R; P459T; T461P; Q174*; R181Q,N,S; G182T,S,N; K185A,R,D,C,E,Q,G,H,I,L,M,N,F,P,S,T,W,Y,V; A186T,S,N,I,V,R; W189T,S,N,Q.
The variants of the invention mentioned above exhibits an alteration in at least one of the following properties relative to the parent xcex1-amylase:
i) improved pH stability at a pH from 8 to 10.5; and/or
ii) improved Ca2+ stability at pH 8 to 10.5, and/or
iii) increased specific activity at temperatures from 10 to 60xc2x0 C., preferably 20-50xc2x0 C., especially 30-40xc2x0 C. Further, details will be described below.
The invention further relates to DNA constructs encoding variants of the invention; to methods for preparing variants of the invention; and to the use of variants of the invention, alone or in combination with other enzymes, in various industrial products or processes, e.g., in detergents or for starch liquefaction.
In a final aspect the invention relates to a method of providing xcex1-amylases with altered pH optimum, and/or altered temperature otimum, and/or improved stability.
Nomenclature
In the present description and claims, the conventional one-letter and three-letter codes for amino acid residues are used. For ease of reference, xcex1-amylase variants of the invention are described by use of the following nomenclature:
Original amino acid(s):position(s):substituted amino acid(s)
According to this nomenclature, for instance the substitution of asparagine for alanine in position 30 is shown as:
ALa30Asn or A30N a deletion of alanine in the same position is shown as:
Ala30* or A30* and insertion of an additional amino acid residue, such as lysine, is shown as:
Ala30AlaLys or A30AK
A deletion of a consecutive stretch of amino acid residues, such as amino acid residues 30-33, is indicated as (30-33)* or xcex94(A30-N33).
Where a specific xcex1-amylase contains a xe2x80x9cdeletionxe2x80x9d in comparison with other xcex1-amylases and an insertion is made in such a position this is indicated as:
*36Asp or *36D for insertion of an aspartic acid in position 36
Multiple mutations are separated by plus signs, i.e.:
Ala30Asp+Glu34Ser or A30N+E34S representing mutations in positions 30 and 34 substituting asparagine and serine for alanine and glutamic acid, respectively.
When one or more alternative amino acid residues may be inserted in a given position it is indicated as
A30N,E or
A30N or A30E
Furthermore, when a position suitable for modification is identified herein without any specific modification being suggested, it is to be understood that any amino acid residue may be substituted for the amino acid residue present in the position. Thus, for instance, when a modification of an alanine in position 30 is mentioned, but not specified, it is to be understood that the alanine may be deleted or substituted for any other amino acid, i.e., any one of:
R,N,D,A,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V.